CTCs ( CIRCULATING TUMOR CELLS )

Some years ago sitting in on a cancer conference just barely keeping my eyes at half mast, the discussion was circumnavigating chemotherapy and the cell cycle. The genetics were being discussed but in a oh so "we do not know what this does yet?" sort of a way, that one could barely perceive the many shades of reality and fiction that mixed in the soup of the unknown. As the conference was winding down an older gentleman, a retired physician, happen to raise his hand and inquire whether it was possible to prevent metastasis by placing patients with cancer on anticoagulants? It was for that time a bold queation. But the only response it was able to garner was a few hrrmphs and a couple of snickers. I don’t quite remember the official answer the speaker rendered but suffice it is to say, it was wanting.

It was a plausible question. In fact it was a well-thought through question. Aside from the dearth of data availability he had questioned the very nature of vascular metastasis.

There were several interesting studies that kept raising the bar of this thought in years to come. The CALGB (Cancer and Leukemia Group B) study done in Stage 1 Lung cancer suggested that when the pleural (lining of the lung) was washed with sterile water and the fluid analyzed, the results showed that 40% of the patients had viable lung cancer cells in the fluid, even though the primary cancer was deep in the lung tissue (parenchyma). Stage I Lung cancer has a 60+% 5-year survival. And  then as in now the question asked, is why should 40% of people with such early resected cancer fall victim to their disease? Or another way to put it; why after all, is it considered stage I and a full, in-toto, resection of the cancer done and yet the patient has only a 60+% chance of survival? One would have to hazard a guess that the resection did not quite reach the R0 state. Something remained rotten in the state. Something surreptitiously ethereal and famously wicked continued to haunt the neighborhood of survival. Something that is in the form of a cancer cell!

Interestingly a Japanese study of Breast Cancer patients showed that almost 40% of the patients in early stage cancer had cancer cells in their bone marrow! These findings gave credence to the fact that even early stage cancers of different origins are already outside of their initial site, meaning they had spread, or more technically speaking "Metastasized."

The concept of Circulating Cells is not a new one. In 1869, yes, we are talking back in the late 19^th Century when Thomas Ashworth observed circulating tumor cells in the blood of a man with metastatic cancer. He surmised that the presence of these cells that looked identical to the ones in the original cancer may “throw some light upon the origins upon the mode of origin of multiple tumours existing in the same person…“One thing is certain, that if they [CTC] came from an existing cancer structure, they must have passed through the greater part of the circulatory system to have arrived at the internal saphena vein of the sound leg.”

Sage musings of a scientific mind, don't you think?

Now comes an even more substantial progression of this thought. Lately and notably there has been a significant impetus in “Circulating Tumor Cells.” These CTCs as they are called are found wading in the blood stream of both early stage and late stage cancers. The early stages have fewer then 5 CTCs in a given quantity (7.5ccs) versus the late stage cancers. But what is interesting is the fact that now we have the capability of detection at a very early level.  In a large multi institutional double-blind prospective clinical trial of breast cancer patients Swaby et al. state; "One hundred and seventy-seven patients with measurable disease had CTCs tested prior to beginning a new palliative treatment regimen for progressive disease, followed by repeat assessment at first follow-up visit approximately 4 weeks later. This landmark trial prospectively identified a CTC cut-off level of ≥5 cells per 7.5 ml of blood to be a reliable identifier of patients at higher risk for disease progression and decreased survival from metastatic breast cancer. Regardless of histology, hormone receptor and HER2/neu status, or whether the patient had recurrent or de novo metastatic disease, those with less than 5 CTCs at baseline, and more importantly, at first follow-up after beginning a new treatment regimen, had superior progression free (PFS) and overall survival (OS) (7 vs 2.1 months [p < 0.001] and 10.1 vs more than 18 months, [p < 0.001], respectively).

These CTCs present an attractive surrogate phenotypic and genotypic markers for disease status in a more dynamic and real-time basis.

Several new studies have been undertaken to be able to decipher their presence and use them as predictor of metastasis and survival. Powell et.al reported:

Patients in a training set with levels of circulating tumor cells equal to or higher than 5 per 7.5 ml of whole blood, as compared with the group with fewer than 5 circulating tumor cells per 7.5 ml, had a shorter median progression-free survival (2.7 months vs. 7.0 months, P<0 .001=".001">

Other related studies have demonstrated with equal aplomb the benefits of CTCs as predictors of response much before radiological data becomes confirmatory, eg. CT scan findings lag behind the drop in the CTCs in the peripheral blood samples.

Left bank shows viable CTC cells, Central bank shows damaged CTCs, Right bank shows sheared cells.

The problems and there are always more then one, are how to confirm that the cells being viewed are indeed cancer cells and if they are cancer cells are they effete (apoptotic) or viable? If they were viable did they have the capability to establish an anchor on to the blood vessels and thence spread into far away organs? If they did have the capability did they have the efficient mode of the EMT (Epithelial Mesenchymal Transition) ability to be able to harness the biochemical powers needed to broach the basement membrane? Additional problems that confound have a molecular basis to them and we will address those by the by.

Circulating Tumor Cells morphologically are distinguishable by their appearance in a majority of cases. Tumor cells have a distinct nuclear/cytoplasmic dys-synchrony, meaning that the nucleus of the cells is a large polyploidal, angry-looking-mushroomy globular structure and the cytoplasm (the surrounding fluid) is small and sometimes negligibly invisible. This is in stark contrast to a normal cell, which has a small well delineated nucleus and a large volume of cytoplasm.

Breast Cancer CTCs

Present State:

Our current modus-operandi is to be able to evaluate the presence of the CTCs. Having done that and shown the presence of such CTCs at a certain limit signifies a poorer prognosis in survival. As Powell et al. and others have shown that there is value in such determinations. Built into this simplicity one still has to undo the large conspiracy of “Is it or is it Not? In other words where the dilemma keeps haunting the researchers, is the ability to accurately decipher that the cell they earmark as a cancer cell in circulation is truly a cancer cell and functional at that!

Colon Cancer CTCs:

(Bonus) Here is one method of isolating the cancer cells from the blood sample (CTCs)

http://www.jove.com/video/4248/rapid-isolation-viable-circulating-tumor-cells-from-patient-blood

Several roads lead to this land of promise; from morphologically deciphering via visual inspection (thus based only on reliance of the observer) to using immuno-histochemical means where chemicals added to the cells can make them more prominent, to using molecular means are all different methodologies currently in vogue.

1.   Since the white cells are the ones that confabulate the issue of recognition between them and the cancer cells. The problem arises when you force the blood through shearing stressors of mechanical corralling in a machine for identification and is related to forces of fluid dynamics. The cellular distortion causes the nucleus to appear as all kinds of monsters even in normal cells and gives the nucleus a "naked" distorted appearance resembling a cancer cell. Thus attempts to verify a good cell from a bad cell has taken many a turn and twist. Using Flourescein labeled antibodies to CD45,  an antigenic determinant for the white cells and also utilizing EP-CAM or Epithelial-Cell adhesion molecules with antibody coated magnetic bead for cancer cells that express Cytokeratin is one way of differentiating between the two: “The procedure enriches the sample for cells expressing the epithelial-cell adhesion molecule with antibody-coated magnetic beads, and it labels the cells with the fluorescent nucleic acid dye 4,2-diamidino-2-phenylindole dihydrochloride. Fluorescently labeled monoclonal antibodies specific for leukocytes (CD45allophycocyan) and epithelial cells (cytokeratin 8,18,19phycoerythrin) are used to distinguish epithelial cells from leukocytes. The identification and enumeration of circulating tumor cells were performed with the use of the CellSpotter Analyzer, a semiautomated fluorescence-based microscopy system that permits computer-generated reconstruction of cellular images. Circulating tumor cells were defined as nucleated cells lacking CD45 and expressing cytokeratin.”

However there is another twist in this particular fate; Aggressive cancer cells lose the ability to project the Ep-CAM on their surface, hence looking for CTC just on the basis of the CD45 and Ep-CAM may falsely eliminate cancer cells and that is exactly what happens in the real world scenarios. For example in the aggressive Inflammatory Breast Cancer or IBC patients the CTC detection based on the two immuno-histochemical technique fails miserably for that specific reason. Maryam B. Lustberg, M.D., MPH, study investigator and assistant professor of internal medicine at The Ohio State University College of Medicine, explains: "Some of the most aggressive forms of cancer, including triple negative breast cancer (TNBC), downregulate EpCAM and have high numbers of EpCAM negative CTCs, cells that would be missed by the most commonly utilized technologies. The results of this study support the theory that there is a heterogeneous population of circulating cells in the blood of cancer patients which may hold clues regarding the metastatic process."

Lung Cancer CTCs:

2. Facing diminishing returns, the incorporation of the genomic makeup of the cancer cell is a more predictable one and is based on mutated genes from the primary source. This has been applied to eliminate the false negativity related to antibody mediated histochemical determinations. Although this may appear to be the answer to the conundrum, it is far from it. Powel et al. again make the following references; “the MagSweeper - a cell purification technology, which gently isolates rare CTCs with high specificity. availed recent advances in microfluidics, isolation protocol does not impact viability and RNA integrity of isolated cells nor gene expression, and as evident here by consistent detection of multiple reference gene transcripts, comprehensive genomic studies on robust subpopulations of cells is greatly facilitated. gene expression profiling of primary tumors and its application in the molecular subtyping of breast cancer has provided a biological framework for defining the clinical heterogeneity of this disease. addressing the issue of how to eliminate the contributions of potentially large numbers of contaminating WBCs to overall gene expression profiles when measuring genes common to both 63% of MagSweeper-captured cells showed robust, non-degraded reference gene expression: of cells with non-degraded reference RNA, 60% were defined as CTCs and 21% expressed the CD45 WBC marker.” So here lies another minor issue; the commonality of genes between normal cells and the cancer cells has also to be taken into consideration. The puzzle pieces thus proliferate from the simple to the very complex. Then one has to address the mutated genes and the lack of commonality of the mutated genes between different metastatic site CTCs. Oh what a web nature weaves!

3. While we are involved in this complicated issue (as it exists – Remember I am just the messenger), here is another dollop of mischief that the cancer cells have in store. The Primary cancer at its initial site has a completely different set of genetic mutations as compared to the cancer that is spreading or one that has already spread but circulating its wealth around. Within the circulating cancer cells there may be a large numbers of populations of metastatic cancer cells from different sites that have different genetic determinants. To elaborate that further; CTCs from the primary cancer cell and others from various metastatic sites (eg. lung, liver, bone etc) all will have differing degrees of mutated genes. So even though we can with clarity state that the cells we are looking at are cancer they will have differing sense of  behavior and create a much different sense of foreboding for the molecular geneticists. “Intriguingly, high levels of PTEN expression in 83% of the CTCs were observed despite the known inverse association between this gene and TGFβ expression. It is possible that repression of this gene by TGFβ requires receptor tyrosine kinase signaling (such as EGFR). which might be compromised as indicated by undetectable EGFR expression in the CTCs in our study.. Consistent with the acquisition of invasive and migratory characteristics is the absence of the cell adhesion protein, CDH1, in migrating cells such as CTCs, as illustrated by our expression data. Ostensibly, systematic implementation of single cell CTC profiling will shed new light on the dynamics of migratory tumor cell biology during metastatic dissemination.”

 Image (Heatmap of single cell gene expression of 87 genes within seven individual cells isolated from three primary tumor-derived (pink: CCdl054, orange: CCdl672, gold: CCdl675), and four metastatic effusion-derived (red: MDA-231 plum: SKBR3, dark green: MCF7, and bright green: T47D) breast cancer cell lines. Yellow indicates high gene expression; gray is median expression; blue indicates low expression; and black represents undetectable expression. All cells showed expected expression patterns. The breast cancer cell lines used represent a spectrum of cell differentiation, e.g., from less differentiated and more mesenchymal/stem cell-like ER-negative (basal-like) cells (MDA-231 and SKBR3) to more differentiated ER-positive (luminal-like) cells represented)

Additional data which should come as no surprise is that CTCs seem to be in abundance in patients with bone metastases and are underrepresented in non-bone metastatic patients. The conjecture here is that bone metastases occur via vascular spread and the marrow of the bone that carry the blood-vessels to and from called vaso-vasorum bring in and take out the CTCs from circulation. Hence the discrepancy of finding CTCs in various forms of stage IV cancers.

Future of CTCs

Okay we seem to be getting better at determining what the CTCs are, how they look like, what their chemical characteristics are and what makes their genetic makeup tick. Looking at how we can possibly manipulate this information to better serve humanity, we will focus on the probabilities ahead.

1. The epitope (antigenic) expression of the cancer cells can be used to target these cancer cells both in the blood stream, metastatic and ultimately also at the primary site  Knowing the cell surface antigens of the various gene-expressions can result in utilizing a targeted set of antibodies directed at these epitopes to neutralize their action in thus suppressing the proliferating cancer cell. For example if gene “A” causes an antigen “X” to be expressed then an antibody “Xa” directed at the antigen “X” will prevent the cells from receiving the signal for growth. That will cut short the cell survival and ultimately put the cancer into a remission. However here too there are some relevant issues that have to be addressed before we  arrive at the Utopian paradise. The cancer cell being visualized may lose some of these expressive antigens and thus their pathways of signal transduction by merely undergoing the shearing stresses mentioned previously. Thus available data may be compromised for relevance. For example Powel et al explain;  “Therapy that targets only one CTC population might not ablate other subpopulations, which may continue to spread and grow.” They also go on to explain that, “Our expression profiling analyses demonstrated that CTC populations are relatively quiescent. Transcript levels of growth factors and their receptors, such as VEGFA, MET, ESR1, EGFR, and HER2 were relatively undetectable in CTCs compared to cancer cell lines. Consequently, expression of downstream effectors involved in cell cycle progression and proliferation such as MYC, ATF3, TERT, RAC1, FOXA1, RRM1, CCNB1, and BIRC5 were significantly diminished in CTCs in contrast to breast cancer cell lines. On the other hand, we found that some CTCs maintained the expression of genes associated with the PI3K-AKT-mTOR cell survival pathway.”

CTC understanding has taken on a whole different meaning since the days of Thomas Ashworth. Oh what a path we have taken, full of glorious achievements and the agony of new found hurdles. Yet each hurdle is a challenge to accept and surpass. Therein lies the wonders of Science and human ingenuity.

One day in not too distant a future, Real-Time analysis will better monitor cancer responses to targeted and non-targeted therapy. These therapeutic responses will be able to predict survival data and in so doing add to the armamentarium of cancer therapy. The resultant decrease in the CTCs will open the door for more selective therapy attacking the unaffected (resistant) cells with specifically directed treatment towards them. Thus with the new bag of tricks we will come ever so close to the ultimate aim of "cure."

Oh and that elderly physician who raised the question about using anticoagulants to prevent or delay metastases, after all may not have been too far off course in his thinking. New data suggests that Heparin interferes with metastasis by mechanisms other than direct interference:  "the anti-metastatic effect of heparin is not a result of its anticoagulant activity but rather its ability to inhibit the interactions between some oligosaccharides present on tumor cells and P-selectin on platelets."

So all in all, the gentleman was correct in his thinking and in his musings too.

Here's to all the thinkers!
Here's to all the doers!

Here’s to human intelligence!

Here’s to human perseverance!

And to all of us; Keep Searching for the Truth!

Circulating Tumor Cells: a Window Into Cancer Metastasis by Daniel Haber - Harvard Medical School/MGH:

http://mit.tv/zh4TXd

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Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LWMM, Hayes DF: Circulating tumor cells, disease progression, and survival in metastatic breast cancer.

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Ashley A. Powell Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines PLoS ONE 7(5): e33788. doi:10.1371/journal.pone.0033788